PFGE in Microbial Molecular Identification

Pulsed field gel electrophoresis (PFGE) is regarded as the "third-generation molecular method" and the "gold standard" for bacterial typing. It is one of the practical methods that enable researchers to detect, identify and classify the genome of micro and macro organisms accurately and sensitively. Creative Biolabs is a leading provider of custom services for live biotherapeutic product (LBP) development. Our team applies decades to offer tailored, flexible solutions for microbial identification matched to your needs.

About PFGE

PFGE permits to resolve mixes of DNA molecules larger than 20 kb and up to 5 Mb in agarose gels. PFGE separates DNA based on the alternating of the two directions in which electric fields are applied, and the ability of the DNA molecule to reorient itself in the new direction in which the electric field is applied. The time consumed by the molecules in the reorientation depends on their sizes. Due to the advantages of PFGE in differentiating bacterial subtypes, PFGE is mainly used in bacterial DNA fingerprinting.

Types of PFGE

  • Contour Clamped Homogeneous Electric Field (CHEF)

CHEF uses the positive electrode to the negative electrode at the angle of orientation 120 degrees. In this system, the size, position, coordination, stability, and continuity of electric fields are precisely controlled, which allows DNA fragments of different sizes to be separated.

  • Field-Inversion Gel Electrophoresis (FIG)

Working with FIGE is easy and simple. FIGE is very popular nowadays because it can separate smaller parts. FIGE offers an acceptable resolution of over 800 Kb.

  • Asymmetric Field Inversion Gel Electrophoresis (AFIGE)

The method is used to detect DNA double-strand breaks (DSBs) by reverse asymmetric field gel electrophoresis.

  • Orthogonal-Field Alternation Gel Electrophoresis (OF AGE)
  • Rotating Gel Electrophoresis (RGE)

RGE has a single electric field and can be separated directly because only one set of electrodes is used.

  • Pulsed-Homogeneous Orthogonal Field Gel Electrophoresis (PHOGE)
  • Programmable Autonomously-Controlled Electrodes (PACE )

General Process of PFGE Analysis

1. Preparation of Bacterial Cells

2. Preparation of Agarose Plugs

3. Plug Lysis

4. Plug Washing

5. Restriction Enzyme Digestion

6. Preparing and Running the Gel

7. Staining and Documentation

8. Data Analysis

A general scheme of the process of preparation of chromosome-sized immobilized DNA in agarose plugs, followed by PFGE analysis.Fig.1 A general scheme of the process of preparation of chromosome-sized immobilized DNA in agarose plugs, followed by PFGE analysis. (Lopez, 2019)

Applications of PFGE

PFGE is used in the following areas:

  • The advent of PFGE techniques for the resolution of large DNA molecules has provided a new analysis approach for bacterial genomes.
  • The PFGE of DNA fragments are obtained using endonuclease to produce a discrete pattern of bands useful for fingerprinting and physical mapping of chromosomes.
  • The PFGE technique helps determine the relationship between different strains of the same species.
  • FGE has proved to be an effective method for estimating genome size and constructing chromosomal maps, as well as characterization of bacterial species.
  • PFGE is a powerful tool for genome characterization.
  • PFGE will play an important role in mapping the human genome.

PFGE is a powerful tool for subtyping bacteria. A good subtyping method should have five main characteristics: typability, reproducibility, discriminability, ease of implementation, and interpretability, and the PFGE method meets all these requirements. If you are developing LBP products and progressing to strain identification and strain typing, you can fully rely on Creative Biolabs’ PFGE service. Our expert scientists and talented technical support team have got you covered all the way. Please contact us for more information.

Reference

  1. Lopez, C.L.; et al. Pulsed field gel electrophoresis: past, present, and future. Analytical biochemistry. 2019, 573: 17-29.

For Research Use Only. Not intended for use in food manufacturing or medical procedures (diagnostics or therapeutics). Do Not Use in Humans.

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